The long-term goal of this research is to resolve molecular and cellular substrates for high-functioning autism. We will develop mouse models for human de novo mutations in ANK2, one of the few genes associated with autism in the absence of intellectual disability, developmental delay, or seizures. ANK2 encodes 220 kDa ankyrin-B (ankB) which is widely expressed, and 440 kDa ankB which is found only in neurons and is localized to unmyelinated axons and growth cones. The distribution of human de novo mutations suggests that mutation of 440 kDa ankB is sufficient to cause high function autism. We have generated mice modeling a human de novo ANK2 frameshift mutation that selectively depletes 440 kDa ankB. We will explore the hypothesis that mutation/deficiency of 440 kDa ankB selectively perturbs both local and long-range neural connectivity through altered axonal function as follows. 1. Resolve the molecular basis for increased axon growth/branching observed in 440 kDa ankB mutant neurons, and establish a cellular assay to evaluate human 440 kDa ankB variants. Neurons cultured from mice lacking 440 kDa ankB exhibit increased axon length and branching. We will explore the roles in this phenomenon of cytoskeletal dynamics and exocytosis. We also will develop a rescue strategy to evaluate functional consequences of human variants of 440 kDa ankB on axon growth/branching. 2. Determine consequences of 440 kDa ankB mutation for establishment of local and long-range neural connectivity during postnatal brain development. 440 kDa ankB-deficient mice exhibit increased cortical synaptogenesis as well as loss of interaction with the cell adhesion molecule L1CAM. Since L1CAM requires ankyrin-binding activity for long distance axon pathfinding, we hypothesize that 440 kDa ankB-deficient neurons will exhibit abnormal axon targeting. We will evaluate local and long-range targeting of axons of during development in 440 kDa ankB-deficient mice using a) diffusion tensor imaging (DTI) b) imaging of axons of cortical neurons sparsely labeled by stereotactic injection of AAV GFP/Cherry constructs; c) analysis of excitatory and inhibitory forebrain synapses during development using immunofluorescence and electrophysiology. 3. Determine a reproducible behavioral phenotype for 440 kDa ankB mutant mice. In collaboration with Drs. William Wetsel (Director of the Duke Mouse Behavioral and Neuroendocrine Analysis Core Facility) and Henry Yin (Duke Department of Psychology and Neuroscience) and co-investigators on this proposal, we will evaluate social behavior, cognition, sensory-motor gating, and repetitive/anxiety-like behavior. In addition, in collaboration with Henry Yin, we will use computer-assisted analysis of 3D imaging to develop an unbiased reproducible behavior profile. These studies of cellular and in vivo consequences of de novo ANK2 mutation promise to provide the first mechanistic insights into high functioning forms of autism.